Hybrid spinach variety 51-335 RZ

ABSTRACT

The present invention relates to a  Spinacia oleracea  seed designated 51-335 RZ, which exhibits late bolting, savoy mature leaves with an ovate shape, and resistance to downy mildew ( Peronospora farinosa  f.sp.  spinaciae ) races Pfs1 to Pfs13 and strain UA4410. The present invention also relates to a  Spinacia oleracea  plant produced by growing the 51-335 RZ seed. The invention further relates to methods for producing the spinach cultivar, represented by spinach variety 51-335 RZ.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSer. No. 61/694,341, filed Aug. 29, 2012.

The foregoing application, and all documents cited therein or duringtheir prosecution (“appin cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated hereinby reference, are hereby incorporated herein by reference, and may beemployed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a new hybrid spinach (Spinacia oleraceaL.) variety which exhibits late bolting, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410.

BACKGROUND OF THE INVENTION

Spinach (Spinacia oleracea L.) is a flowering vegetable plant belongingto the family Amaranthaceae. It is native to Southwestern and CentralAsia, but today spinach is cultivated worldwide, mainly in temperateregions. Spinach plants are cultivated for their highly nutritiousleaves. The leaves are extremely rich in antioxidants, a good source ofvitamins such as A, B6, C, E, K and folate, and minerals such ascalcium, iron, magnesium and potassium.

The edible part of the spinach plant is a compact rosette shape ofleaves attached to a short stem. The leaves are produced during thefirst stage of the plant's life cycle, the vegetative rosette stage. Thesecond stage is the flowering stage, or the bolting stage. When boltingoccurs, there is growth of an elongated stalk with flowers growing fromwithin the main stem of the plant. Once the plant has reached thebolting stage, it is no longer possible to harvest marketable leaves.The plant will allocate its resources to flowering instead of leafproduction, which will ultimately cause the leaves to wither. Fastbolting is thus a very undesired trait in the production of leafyvegetables such as spinach. Therefore, slower bolting is very muchpreferred by growers in order to optimize yield.

There are three basic types of spinach, namely savoy, semi-savoy, andsmooth. The savoy type spinach have dark green, crinkly and curlyleaves. The semi-savoy type spinach is a hybrid variety with slightlycrinkled leaves. Smooth type spinach has broad, smooth leaves.

The leaves of a spinach plant are usually sold loose, bunched, inpre-packaged bags, canned or frozen. Each of these products requires itsown type of leaves. The smooth and some of the semi-savoy varieties arepredominantly used in processed products such as canned or frozenspinach, while the semi-savoy and savoy types are especially used forthe fresh market. Smooth leaf varieties are better suited for processingpurposes than semi-savoy and savoy varieties, because they are easier toclean and prepare for canning and freezing. Savoy leaf varieties, on theother hand, are preferred in the fresh market because the leaves packlooser than the smooth leaf varieties and are less likely to wilt orturn yellow.

Downy mildew is probably the most widespread and potentially destructiveglobal disease of spinach. The causal agent of downy mildew on variousAmaranthaceae, including spinach, is regarded as a single species,Peronospora farinosa. In particular, Peronospora farinosa f. sp.spinaciae infects spinach. Initial symptoms of downy mildew consist ofdull to bright yellow necrotic lesions that appear on the leaves ofinfected spinach plants. With time the lesions can enlarge and becometan and dry. The infection can spread very rapidly, and it can occurboth in glasshouse cultivation and in soil cultivation, resulting inwidespread crop damage. In addition to the loss of quality of the leavesdue to the lesions, downy mildew can also cause breakdown and rot of theinfected leaves if they packaged in bags and cartons.

The optimal temperature for formation and germination of P. farinosa f.sp. spinaciae spores is 9 to 12° C., and it is facilitated by a highrelative humidity. When spores are deposited on a humid leaf surfacethey can readily germinate and infect the leaf. Fungal growth is optimalbetween 8 and 20° C. and a relative humidity of ≧80%, and within 6 and13 days after infection mycelium growth can be observed. Oospores of P.farinosa can survive in the soil for up to 3 years, or as mycelium inseeds or living plants. Although some fungicide treatments may beeffective, they can be costly and may cause ecological or pollutionconcerns, and may not be desired by organic farmers.

In recent years various resistance genes have been identified thatprovide spinach plants with a resistance against downy mildew. However,it has been observed that previously resistant spinach cultivars canagain become susceptible to the fungus. Investigations revealed that thecultivars themselves had not changed, and that the loss of downy mildewresistance must therefore be due to P. farinosa overcoming theresistance in these spinach cultivars. The downy mildew races (alsocalled physios, isolates, or strains) that were able to infect resistantspinach cultivars were collected in a differential reference set, whichcan be used to test spinach cultivars for resistance. The differentialset may comprise a series of spinach cultivars (hybrids) that havedifferent resistance patterns to the currently identified pathogenicraces.

Currently there are 13 officially recognised races of Peronosporafarinosa f. sp. spinaciae, designated races pfs1 to pfs13 (Irish et al.Phytopathol. Vol. 98 number pg. 894-900, 2008; Plantum NL (Dutchassociation for breeding, tissue culture, production and trade of seedand young plants) press release, “Denomination of Pfs: 12, a new race ofdowny mildew in spinach”, Feb. 28, 2011; Plantum NL (Dutch associationfor breeding, tissue culture, production and trade of see and youngplants) press release, “Benoeming van fysio Pfs: 13, een nieuwe fysiovan valse meeldauw in spinazie”, Aug. 2, 2011. Recently another newPeronospora isolate has been identified, which has not yet received anofficial “Pfs” number, but which is assumed to become known as Pfs14 inthe near future: UA4410. This isolate is publicly available from theDepartment of Plant Pathology, University of Arkansas, Fayetteville,Ark. 72701, USA, along with the 13 officially recognized Pfs races. Seee.g., Correll and Koike, “Race diversity and the biology of spinachdowny mildew pathogen”, Mar. 15, 2011 CLRGB Progress Report, and Feng etal, “Characterization of new races and novel strains of the spinachdowny mildew pathogen Peronospora farinose f. sp. spinaciae”, 2011International Spinach Conference Oct. 3-4, 2011, Amsterdam andEnkhuizen, The Netherlands (Abstract in Updated Program Sep. 26, 2011).

Newly identified Peronospora races can break the resistance of manyspinach varieties that are currently used commercially worldwide, andthey thus pose a serious threat to the productivity of the spinachindustry. For this reason new resistance genes are very valuable assets.In order to confer a resistance that is as broad as possible, i.e. thatconfers resistance to as many Pfs races as possible, preferable to allknown Pfs races, it is very useful to be able to stack differentresistance genes against Peronospora infection in spinach. This isachieved by stacking various resistance genes, which have overlappingresistance patterns. In this way it becomes more difficult for thepathogen to overcome the resistance. Such a combination of differentresistance genes on one gene segment is also highly desirable. It ismuch easier if the resistance genes can be inherited as a singledominant loci, because in that case the resistance pattern that isconferred by the dominant resistance gene cannot segregate in theprogeny of the cross, and will always inherit as one single set ofresistances to various pathogenic races. One such resistance gene,designated R6, is a single dominantly inherited resistance gene inspinach that confers resistance to Peronospora farinosa f. sp. spinaciaeraces Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs9, Pfs11, Pfs12, Pfs13, andthe most recently identified isolate UA4410(See generally EP applicationNo. 11187288).

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There is a need for a hybrid spinach variety which exhibits acombination of traits including late bolting, savoy mature leaves withan ovate shape, and resistance to downy mildew (Peronospora farinosaf.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410.

The present invention addresses this need by providing a new type ofspinach (Spinacia oleracea) variety, designated 51-335 RZ. Spinachcultivar 51-335 RZ exhibits a combination of traits including latebolting, savoy mature leaves with an ovate shape, and resistance todowny mildew (Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13and strain UA4410.

The present invention provides seeds of spinach cultivar 51-335 RZ,which have been deposited with the National Collections of Industrial,Marine and Food Bacteria (NCIMB) in Bucksburn, Aberdeen AB21 9YA,Scotland, UK and have been assigned NCIMB Accession No. 41980.

In one embodiment, the invention provides a spinach plant which mayexhibit a combination of traits including late bolting, savoy matureleaves with an ovate shape, and resistance to downy mildew (Peronosporafarinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410,representative seed of which have been deposited under NCIMB AccessionNo. 41980.

In one embodiment, the invention provides a spinach plant which mayexhibit a combination of traits including late bolting, elliptic firstfoliage leaves, savoy mature leaves with an ovate shape, and resistanceto downy mildew (Peronospora farinosa f.sp. spinaciae) races Pfs1 toPfs13 and strain UA4410, representative seed of which have beendeposited under NCIMB Accession No. 41980.

In one embodiment, the invention provides a spinach plant which mayexhibit a combination of traits including late bolting, elliptic firstfoliage leaves with a round pointed tip, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410, representative seed ofwhich have been deposited under NCIMB Accession No. 41980.

In one embodiment, the invention provides a spinach plant designated51-335 RZ, representative seed of which have been deposited under NCIMBAccession No. 41980.

In an embodiment of the present invention, there also is provided partsof a spinach plant of the invention, which may include parts of aspinach plant exhibiting a combination of traits including late bolting,savoy mature leaves with an ovate shape, and resistance to downy mildew(Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strainUA4410, or parts of a spinach plant having any of the aforementionedresistance(s) and a combination of traits including one or moremorphological or physiological characteristics tabulated herein,including parts of hybrid spinach variety 51-335 RZ, wherein the plantparts are involved in sexual reproduction, which include withoutlimitation, microspores, pollen, ovaries, ovules, embryo sacs or eggcells and/or wherein the plant parts are suitable for vegetativereproduction, which include, without limitation, cuttings, roots, stems,cells or protoplasts and/or wherein the plant parts are tissue cultureof regenerable cells in which the cells or protoplasts of the tissueculture are derived from a tissue such as, for example and withoutlimitation, leaves, pollen, embryos, cotyledon, hypocotyls, meristematiccells, roots, root tips, anthers, flowers, seeds or stems. The plants ofthe invention from which such parts may come from include those whereinrepresentative seed of which has been deposited under NCIMB AccessionNo. 41980, or hybrid spinach variety or cultivar designated 51-335 RZ,as well as seed from such a plant, plant parts of such a plant (such asthose mentioned herein) and plants from such seed and/or progeny of sucha plant, advantageously progeny exhibiting such combination of suchtraits, each of which, is within the scope of the invention.

In another embodiment there is a plant grown from seeds, representativeseed of which having been deposited under NCIMB Accession No. 41980. Ina further embodiment there is a plant regenerated from theabove-described plant parts or regenerated from the above-describedtissue culture. Advantageously such a plant may have morphologicaland/or physiological characteristics of hybrid spinach variety 51-335 RZand/or of a plant grown from seed, representative seed of which havingbeen deposited under NCIMB Accession No. NCIMB 41980—including withoutlimitation such plants having all of the morphological and physiologicalcharacteristics of hybrid spinach variety 51-335 RZ and/or of plantgrown from seed, representative seed of which having been depositedunder NCIMB Accession No. NCIMB 41980. Advantageously, such a plantdemonstrates the traits of late bolting, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410. Accordingly, in stilla further embodiment, there is provided a spinach plant having all ofthe morphological and physiological characteristics of hybrid spinachvariety 51-335 RZ, representative seed of which having been depositedunder NCIMB Accession No. 41980. Such a plant may be grown from theseeds, regenerated from the above-described plant parts, or regeneratedfrom the above-described tissue culture. A spinach plant having any ofthe aforementioned resistance(s), and one or more morphological orphysiological characteristics recited or tabulated herein, and a spinachplant advantageously having all of the aforementioned resistances andthe characteristics recited and tabulated herein, are preferred. Partsof such plants—such as those plant parts above-mentioned—are encompassedby the invention.

In one embodiment, there is provided progeny of spinach cultivar 51-335RZ produced by sexual or vegetative reproduction, grown from seeds,regenerated from the above-described plant parts, or regenerated fromthe above-described tissue culture of the spinach cultivar or a progenyplant thereof, representative seed of which having been deposited underNCIMB Accession No. 41980. The progeny may have any of theaforementioned resistance(s), and one or more morphological orphysiological characteristics recited or tabulated herein, and a spinachplant advantageously having all of the aforementioned resistances andthe characteristics recited and tabulated herein, are preferred.Advantageously, the progeny demonstrate the traits of late bolting,savoy mature leaves with an ovate shape, and resistance to downy mildew(Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strainUA4410.

Progeny of the hybrid spinach variety 51-335 RZ may be modified in oneor more other characteristics, in which the modification is a result of,for example and without limitation, mutagenesis or transformation with atransgene.

In still another embodiment, the present invention provides progeny ofspinach cultivar 51-335 RZ produced by sexual or vegetativereproduction, grown from seeds, regenerated from the above-describedplant parts, or regenerated from the above-described tissue culture ofthe spinach cultivar or a progeny plant thereof, in which theregenerated plant shows a combination of traits including late bolting,savoy mature leaves with an ovate shape, and resistance to downy mildew(Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strainUA4410. The progeny may have any of the aforementioned resistance(s),and one or more morphological or physiological characteristics recitedor tabulated herein, and a progeny plant advantageously having all ofthe aforementioned resistances and the characteristics recited andtabulated herein, is preferred. Advantageously, the progeny demonstratethe traits of late bolting, savoy mature leaves with an ovate shape, andresistance to downy mildew (Peronospora farinosa f.sp. spinaciae) racesPfs1 to Pfs13 and strain UA4410.

In another embodiment the invention relates to a method of producing aninbred spinach plant derived from a plant of the invention of whichrepresentative seed has been deposited under NCIMB Accession No. NCIMB41980, which may comprise of the steps: a) preparing a progeny plantderived from hybrid spinach variety 51-335 RZ by crossing the plant of aspinach plant exhibiting a combination of traits including late bolting,savoy mature leaves with an ovate shape, and resistance to downy mildew(Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strainUA4410, representative seed of which have been deposited under NCIMBAccession No. 41980 with a second spinach plant and selecting plantsexhibiting at least one or more traits of late bolting, savoy matureleaves with an ovate shape, and resistance to downy mildew (Peronosporafarinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410 as aprogeny plant; b) crossing the progeny plant with itself or a secondspinach plant to produce a seed of a progeny plant of a subsequentgeneration; c) growing a progeny plant of a subsequent generation fromsaid seed and crossing the progeny plant of a subsequent generation withitself or a second spinach plant and selecting plants exhibiting atleast one of the traits of late bolting, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410; and d) repeating stepb) or c) for at least 1 more generation to produce an inbred spinachplant derived from the hybrid spinach variety 51-335 RZ that has atleast one of the traits of late bolting, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410, and advantageously atleast the resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410. Of course, theselections may be for plants having or exhibiting or carrying (e.g.recessively) any of the aforementioned resistance(s), and one or moremorphological or physiological characteristics recited or tabulatedherein, and advantageously having or exhibiting or carrying all of theaforementioned resistances and the characteristics recited and tabulatedherein, are preferred. More advantageously, the progeny and the productof the method demonstrate the traits of late bolting, savoy matureleaves with an ovate shape, and resistance to downy mildew (Peronosporafarinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410.

The invention even further relates to a method of producing spinachwhich may comprise: (a) cultivating to the vegetative plant stage aplant of hybrid spinach variety 51-335 RZ, representative seed of whichhaving been deposited under NCIMB Accession No. NCIMB 41980, (b)harvesting spinach leaves from the plant, and (c) optionally using theharvested spinach leaves as a fresh vegetable or (d) optionally usingthe harvested spinach leaves as a processed food. The harvested spinachleaves of step (c) or step (d) may optionally have undergone one or moreprocessing steps. Such a processing step might comprise but is notlimited to any one of the following treatments or combinations thereof:cutting, washing, blanching, cooking, steaming, baking, pasteurizing,canning, or flash-freezing. The processed form that is obtained is alsopart of this invention. The invention further comprehends packaging thespinach leaves in fresh or processed form.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, and “comprising” and the like(e.g., “includes”, “included”, “including”, “contains”, “contained”,“containing”, “has”, “had”, “having”, etc.) can have the meaningascribed to them in US Patent law, i.e., they are open ended terms. Forexample, any method that “comprises,” “has” or “includes” one or moresteps is not limited to possessing only those one or more steps and alsocovers other unlisted steps. Similarly, any plant that “comprises,”“has” or “includes” one or more traits is not limited to possessing onlythose one or more traits and covers other unlisted traits. Similarly,the terms “consists essentially of” and “consisting essentially of” havethe meaning ascribed to them in US Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. See also MPEP §2111.03. In addition, the term “about” is usedto indicate that a value includes the standard deviation of error forthe device or method being employed to determine the value.

These and other embodiments are disclosed or are obvious from andencompassed by the following Detailed Description.

DEPOSIT

The Deposit with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on May 28, 2012, under depositaccession number NCIMB 41980 was made pursuant to the terms of theBudapest Treaty. Upon issuance of a patent, all restrictions upon thedeposit will be removed, and the deposit is intended to meet therequirements of 37 CFR §§1.801-1.809. The deposit will be irrevocablyand without restriction or condition released to the public upon theissuance of a patent. The deposit will be maintained in the depositoryfor a period of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawing, in which:

FIG. 1 is an illustration of seven different leaf shapes from plants atprime market stage grown under optimal conditions.

FIG. 2 is an illustration of three different leaf tip shapes from plantsat the first foliage and the prime market stage grown under optimalconditions.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of a new hybrid spinach variety herein referred toas hybrid spinach variety 51-335 RZ. 51-335 RZ is a hybrid plant varietythat is uniform and distinct from other such hybrids, and may be stablyproduced after a cycle of reproduction.

There are numerous steps in the development of any novel, plant withdesirable characteristics. Selection of traits is a very importantaspect of plant breeding. Once desirable traits are identified, theplants with those desirable traits are crossed in order to recombine thedesirable traits and through selection, varieties or parent lines aredeveloped. The goal is to combine in a single variety or hybrid animproved combination of desirable traits from the parent plant. Theseimportant traits may include but are not limited to higher yield, fieldperformance, fruit and agronomic quality such as fruit shape, color andlength, resistance to diseases and insects, and tolerance to drought andheat.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F1 hybrid cultivar, pure linecultivar, etc.). Popular selection methods commonly include but are notlimited to pedigree selection, modified pedigree selection, massselection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach isused extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

The development of commercial spinach hybrids relates to the developmentof spinach parental lines, the crossing of these lines, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichlines are developed by selfing and selection of desired phenotypes. Thenew lines are crossed with other lines and the hybrids from thesecrosses are evaluated to determine which have the desirablecharacteristics.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F1. An F2 population is produced by selfing one or severalF1s or by intercrossing two F1s (sib mating). Selection of the bestindividuals is usually begun in the F2 population; then, beginning inthe F3, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F4 generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F6 and F7), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections may be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Other methods of breeding may also relate to the single-seed descentprocedure which refers to planting a segregating population, harvestinga sample of one seed per plant, and using the one-seed sample to plantthe next generation. When the population has been advanced from the F2to the desired level of inbreeding, the plants from which lines arederived will each trace to different F2 individuals. The number ofplants in a population declines each generation due to failure of someseeds to germinate or some plants to produce at least one seed. As aresult, not all of the F2 plants originally sampled in the populationwill be represented by a progeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant may alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; thesetechniques include but are not limited to Isozyme Electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length polymorphisms(AFLPs), Simple Sequence Repeats (SSRs—which are also referred to asMicrosatellites), and Single Nucleotide Polymorphisms (SNPs)

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci may be routinely used and more alleles permarker locus may be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers may also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest may be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers may also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It may also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intospinach varieties. Mutations that occur spontaneously or areartificially induced may be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates may be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding may be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company, 1993.

The production of double haploids may also be used for the developmentof homozygous lines in a breeding program. Double haploids are producedby the doubling of a set of chromosomes from a heterozygous plant toproduce a completely homozygous individual. For example, see Wan et al.,Theor. Appl. Genet., 77:889-892, 1989.

The spinach plant of the invention may be arrived at through crossing ofinbred lines or through selection of the disclosed desirablecharacteristics by any of the breeding the selection methods mentionedabove.

The parents of hybrid spinach variety 51-335 RZ were developed asfollows: The mother is a spinach line made from pedigree selection fromno. 06.99713. In total three selection, crossing and selfing cycles, andtwo rounds of mass generation and selection were performed. The fatherline of 51-335 RZ is made from pedigree selection from no. 09.79121,obtained by four selection and inbreeding cycles, and two rounds of massgeneration and selection. Crossing the described mother and fatherinbred spinach lines with one another will yield uniform F1 hybridprogeny plants. Table 1 shows the pedigree scheme of the mother line,Table 2 shows the pedigree scheme of the father line of hybrid spinachline 51-335 RZ.

TABLE 1 Breeding history of the mother line of 51-335 RZ. Year Year 1 F1Pedigree selection from 06.99713 Year 2 S1 F1 generation grown Year 3 S2F1 generation grown Year 4 M1 S2 F1 generation grown (in mass) Year 8 M2S2 F1 generation grown (in mass)

TABLE 2 Breeding history of the father line of 51-335 RZ. Year Year 1 F1Pedigree selection from 09.79121 Year 2 S1 F1 generation grown Year 2 S2F1 generation grown (in mass) Year 3 M1 S2 F1 generation grown Year 3 S1M1 S2 F1 generation grown Year 4 M1 S1 M1 S3 S2 F1 generation grown (inmass)

In one embodiment, a plant of the invention has all the morphologicaland physiological characteristics of spinach variety 51-335 RZ. Thesecharacteristics of a spinach plant of the invention, e.g. variety 51-335RZ, are summarized and compared to its closest publicly availablevariety in Table 3.

The information presented in Table 3 was determined in trial experimentsin accordance with the Exhibit C Form (Form Approved OMB NO 0581-0055)provided by the United States Department of Agriculture Plant VarietyProtection Office. The terminology used in these tables is the officialterminology found in the Exhibit C Form as of the filing date, and isthus clear for a person skilled in the art.

TABLE 3 Physiological and morphological characteristics of 51-335 RZ incomparison with closest known variety “Toucan”. Character 51-335 RZToucan Ploidy Diploid Diploid Plant (Prime market stage): Habit FlatFlat Size Medium Medium Spread (cm) 23 20 Height (cm) 7 5 Seedlingcotyledon Width (mm) 7 7 Length (mm) 49 52 Tip Pointed Pointed Colorchart RHS 146A RHS 146A Leaf (First foliage leaves) Shape Elliptic BroadOvate Base Lobed Lobed Tip Round pointed Round Margin Slightly curledSlightly curled Upper surface color chart RHS 146A RHS 146A Lowersurface color chart RHS 146B RHS 146B Leaf (Prime market stage) SurfaceSavoy Smooth Shape Ovate Circular Base Lobed Straight Tip Round RoundMargin Slightly curled Slightly curled Upper surface color chart RHS146A RHS 146A Lower surface color chart RHS 146B RHS 146 B Luster GlossyDull Blade size Large Medium Blade lobing Lobed Not lobed Petiole colorLight green Light yellow Color chart RHS 146B RHS 146B Petiole redpigmentation Absent Absent Petiole length to blade (cm) 2.6 4.4 Petiolediameter (mm) 4 5 Bolting Late to very late Very late (10% plantsbolting)

Aside from the morphological and physiological characteristics mentionedin Table 3, a plant of the invention also exhibits resistance to downymildew (Peronospora farinosa f. sp. spinaciae). As used herein,resistance to Peronospora farinosa f. sp. spinaciae is defined as theability of a plant to resist infection by each of the various races Pfs1to Pfs13, and the most recently identified isolate UA4410 in all stagesbetween the seedling stage and the harvestable prime market stage.

The resistance to downy mildew infection was assayed as described byIrish et al. (2008; Phytopathol. 98: 894-900; incorporated herein byreference), using a differential set. Resistance is tested byinoculating plants at the true leaf stage, and observing symptoms ofchlorosis and sporulation 7 days later.

As used herein resistance to Peronospora farinosa f sp. spinaciae racesPfs1 to Pfs13, and isolate UA4410, may be conferred by one or moreresistance genes or a combination thereof, but most preferably bystacking with the single dominantly inherited R6 gene (See generally EPapplication No. 11187288). The R6 gene is a single dominant geneconferring resistance against Peronospora farinosa f sp. spinaciaestrains Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs9, Pfs11, Pfs12Pfs13, andthe most recently identified isolate UA4410. In table 4 the resistancepattern conferred by the R6 gene is compared with the resistance patternas present in publicly available varieties. Variety Lion is resistant tothe same Peronospora farinosef. sp. spinaciae races, however thisresistance is conferred by multiple genes. This is demonstrated byincluding the parents of hybrid variety Lion. None of the parents isresistant to all of the Peronospora farinosef sp. spinaciaeraces Pfs1,Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs9, Pfs11, Pfs12, Pfs13 and isolateUA4410, indicative that the genetic basis for the resistance pattern ofLion is multigenic, and caused by the stacking of at least tworesistance genes. Therefore, it may be concluded that hybrid varietyLion does not comprise the single dominant R6 gene conferring resistanceto races Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs9, Pfs11, Pfs12,Pfs13,and isolate UA4410.

Next to the deposit of hybrid spinach variety 51-335 RZ, a spinach plantwhich may comprise the R6 gene as present in hybrid variety 51-335 RZhas been deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on Jul. 26, 2011, under depositaccession number NCIMB 41857.

TABLE 4 Lion male Lion female Race Viroflay Resistoflay CaliflayClermont Campania Boeing Lion Lazio Whale Polka Pigeon R6 parent parentPfs: 1 + − − − − − − − − − − − − − Pfs: 2 + − + − − − − − − − − − − +Pfs: 3 + + − − − − − − − − − − − − Pfs: 4 + + + − − − − − − + − − − +Pfs: 5 + + − + − − − − − − − − − − Pfs: 6 + + + + + − − − − + − − − +Pfs: 9 + + − + + − − − − − − − − − Pfs: 11 + + − + − − − + − − − − − −Pfs12 + + − + + + − + − − − − + − Pfs13 + + (+) + (+) − − + + (+) − − −(+) UA4410 + + − + + + − + − − + − + −

As used herein, the leaf surface is by visual comparison to standardvarieties. Three different leaf surface types are recognised: smooth,semi-savoy, or savoy. Leaf surface is determined by comparison tostandard varieties Viroflay, Northland, and Virginia Savoy. Viroflay hassmooth leaves, Northland has semi-savoy leaves, while Virginia Savoy hassavoy leaves. The leaf surface of hybrid spinach variety 51-335 RZ issavoy which is similar to the Virginia Savoy variety.

As used herein, bolting is defined in concordance with the definition onthe UPOV TG/55/7 Form as the time it requires for 15% of the plants tobegin to have a central flowering stem appear through the stretching ofthe internodes for spring sown crops. Bolting is determined bycomparison to standard varieties Figo, Maracas, Bandola, Viroflay,Matador, Monnopa, Grappa, Medania, Revolver, Chica and Lavewa. Figo andMaracas are very early bolting varieties; Bandola and Viroflay are earlybolting varieties; Matador and Monnopa are medium bolting varieties;Grappa, Medania and Revolver are late bolting varieties and Chica andLavewa are very late bolting varieties. Hybrid spinach variety 51-335 RZhas a timing of bolting that is similar to that of Grappa, Medania andRevolver, and is therefore considered a late bolting variety.

Bolting may also be defined using the Exhibit C Form provided by theUnited States Department of Agriculture Plant Variety Protection Office,as the time it requires for 10% of the plants to begin to have a centralflowering stem appear through the stretching of the internodes. Boltingis determined by comparison to standard varieties Dixie Market, LongStanding Bloomsdale and Norgreen. Dixie Market is a early boltingvariety, Long Standing Bloomsdale is a medium bolting variety andNorgreen is a late bolting variety. Hybrid spinach variety 51-335 RZ hasa timing of bolting that is later than that of variety Norgreen, and maytherefore under the exhibit C definition be considered as a late to verylate bolting variety (see Table 3).

The standard varieties Dixie Market, Long Standing Bloomsdale andNorgreen, used for comparison on the Exhibit C form, are all old spinachvarieties. Old late bolting varieties are by today's standards mediumbolting varieties. The Exhibit C early bolting variety Dixie Marketwould be defined as being a very early bolting variety using the UPOVTG/55/7 Form definition. The Exhibit C medium bolting variety LongStanding Bloomsdale would be defined as being an early bolting varietyusing the UPOV TG/55/7 Form definition. The Exhibit C late boltingvariety Norgreen would be defined as being a medium bolting varietyusing the UPOV TG/55/7 Form definition.

As used herein, the leaf shape terminology is in concordance with thatused on the Exhibit C Form provided by the United States Department ofAgriculture Plant Variety Protection Office and are shown in FIG. 1.

As used herein, the leaf tip shape terminology is in concordance withthat used on the Exhibit C Form provided by the United States Departmentof Agriculture Plant Variety Protection Office and are shown in FIG. 2.

Embodiments of the invention advantageously have one or more, and mostadvantageously all, of these characteristics.

In an embodiment, the invention relates to spinach plants that has allthe morphological and physiological characteristics of the invention andhave acquired said characteristics by introduction of the geneticinformation that is responsible for the characteristics from a suitablesource, either by conventional breeding, or genetic modification, inparticular by cisgenesis or transgenesis. Cisgenesis is geneticmodification of plants with a natural gene, coding for an (agricultural)trait, from the crop plant itself or from a sexually compatible donorplant. Transgenesis is genetic modification of a plant with a gene froma non-crossable species or a synthetic gene.

Just as useful traits that may be introduced into a hybrid bybackcrossing the trait into one or both parents, useful traits may beintroduced directly into the plant of the invention, being a plant ofhybrid spinach variety 51-335 RZ, by genetic transformation techniques;and, such plants of hybrid spinach variety 51-335 RZ that haveadditional genetic information introduced into the genome or thatexpress additional traits by having the DNA coding there for introducedinto the genome via transformation techniques, are within the ambit ofthe invention, as well as uses of such plants, and the making of suchplants.

Genetic transformation may therefore be used to insert a selectedtransgene into the plant of the invention, being a plant of hybridspinach variety 51-335 RZ or may, alternatively, be used for thepreparation of transgenes which may be introduced by backcrossing.Methods for the transformation of plants, including spinach, are wellknown to those of skill in the art.

Vectors used for the transformation of spinach cells are not limited solong as the vector may express an inserted DNA in the cells. Forexample, vectors which may comprise promoters for constitutive geneexpression in spinach cells (e.g., cauliflower mosaic virus 35Spromoter) and promoters inducible by exogenous stimuli may be used.Examples of suitable vectors include pBI binary vector. The “spinachcell” into which the vector is to be introduced includes various formsof spinach cells, such as cultured cell suspensions, protoplasts, leafsections, and callus. A vector may be introduced into spinach cells byknown methods, such as the polyethylene glycol method, polycationmethod, electroporation, Agrobacterium-mediated transfer, particlebombardment and direct DNA uptake by protoplasts. To effecttransformation by electroporation, one may employ either friabletissues, such as a suspension culture of cells or embryogenic callus oralternatively one may transform immature embryos or other organizedtissue directly. In this technique, one would partially degrade the cellwalls of the chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wound tissues in a controlled manner.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate. An illustrativeembodiment of a method for delivering DNA into plant cells byacceleration is the Biolistics Particle Delivery System, which may beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a surface covered with targetspinach cells. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge. Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually any plant species, including a plantof spinach variety 51-335 RZ.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA may be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast.Agrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Moreover, advances in vectors for Agrobacterium-mediated gene transferhave improved the arrangement of genes and restriction sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. The vectors have convenientmulti-linker regions flanked by a promoter and a polyadenylation sitefor direct expression of inserted polypeptide coding genes.Additionally, Agrobacterium containing both armed and disarmed Ti genesmay be used for transformation. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene locustransfer. The use of Agrobacterium-mediated plant integrating vectors tointroduce DNA into plant cells, including spinach plant cells, is wellknown in the art (See, e.g., U.S. Pat. Nos. 7,250,560 and 5,563,055).

Transformation of plant protoplasts also may be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for spinach plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, a tandemly duplicated version of the CaMV 35Spromoter, the enhanced 35S promoter (P-e35S), the nopaline synthasepromoter, the octopine synthase promoter, the figwort mosaic virus(P-FMV) promoter (see U.S. Pat. No. 5,378,619), an enhanced version ofthe FMV promoter (P-eFMV) where the promoter sequence of P-FMV isduplicated in tandem, the cauliflower mosaic virus 19S promoter, asugarcane bacilliform virus promoter, a commelina yellow mottle viruspromoter, the promoter for the thylakoid membrane proteins from spinach(psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS) (see U.S. Pat. No.7,161,061), the CAB-1 promoter from spinach (see U.S. Pat. No.7,663,027), the promoter from maize prolamin seed storage protein (seeU.S. Pat. No. 7,119,255), and other plant DNA virus promoters known toexpress in plant cells. A variety of plant gene promoters that areregulated in response to environmental, hormonal, chemical, and/ordevelopmental signals may be used for expression of an operably linkedgene in plant cells, including promoters regulated by (1) heat, (2)light (e.g., pea rbcS-3A promoter, maize rbcS promoter, or chlorophylla/b-binding protein promoter), (3) hormones, such as abscisic acid, (4)wounding (e.g., wunl, or (5) chemicals such as methyl jasmonate,salicylic acid, or Safener. It may also be advantageous to employorgan-specific promoters.

Exemplary nucleic acids which may be introduced to the spinach varietyof this invention include, for example, DNA sequences or genes fromanother species, or even genes or sequences which originate with or arepresent in spinach species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA may include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a plant of spinach variety 51-335 RZ.Non-limiting examples of particular genes and corresponding phenotypesone may choose to introduce into a spinach plant include one or moregenes for insect tolerance, pest tolerance such as genes for fungaldisease control, herbicide tolerance, and genes for quality improvementssuch as yield, nutritional enhancements, environmental or stresstolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s).

Alternatively, the DNA coding sequences may affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms. The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product. Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention. (See also U.S. Pat. No.7,576,262, “Modified gene-silencing RNA and uses thereof”)

U.S. Pats. Nos. 7,230,158, 7,122,720, 7,081,363, 6,734,341, 6,503,732,6,392,121, 6,087,560, 5,981,181, 5,977,060, 5,608,146, 5,516,667, eachof which, and all documents cited therein are hereby incorporated hereinby reference, consistent with the above INCORPORATION BY REFERENCEsection, are additionally cited as examples of U.S. patents that mayconcern transformed spinach and/or methods of transforming spinach orspinach plant cells, and techniques from these US patents, as well aspromoters, vectors, etc., may be employed in the practice of thisinvention to introduce exogenous nucleic acid sequence(s) into a plantof spinach variety 51-335 RZ (or cells thereof), and exemplify someexogenous nucleic acid sequence(s) which may be introduced into a plantof spinach variety 51-335 RZ (or cells thereof) of the invention, aswell as techniques, promoters, vectors etc., to thereby obtain furtherplants of spinach variety 51-335 RZ, plant parts and cells, seeds, otherpropagation material harvestable parts of these plants, etc. of theinvention, e.g. tissue culture, including a cell or protoplast, such asan embryo, meristem, cotyledon, pollen, leaf, anther, root, root tip,pistil, flower, seed or stalk.

The invention further relates to propagation material for producingplants of the invention. Such propagation material may comprise interalia seeds of the claimed plant and parts of the plant that are involvedin sexual reproduction. Such parts are for example selected from thegroup consisting of seeds, microspores, pollen, ovaries, ovules, embryosacs and egg cells. In addition, the invention relates to propagationmaterial which may comprise parts of the plant that are suitable forvegetative reproduction, for example cuttings, roots, stems, cells,protoplasts.

According to a further aspect thereof the propagation material of theinvention may comprise a tissue culture of the claimed plant. The tissueculture may comprise regenerable cells. Such tissue culture may bederived from leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems(See generally U.S. Pat. No. 7,041,876 on spinach being recognized as aplant that may be regenerated from cultured cells or tissue).

Also, the invention comprehends methods for producing a seed of a“51-335 RZ”-derived spinach plant which may comprise (a) crossing aplant of spinach variety 51-335 RZ, representative seed of which havingbeen deposited under NCIMB Accession No. NCIMB 41980, with a secondspinach plant, and (b) whereby seed of a “51-335 RZ”-derived spinachplant form (e.g., by allowing the plant from the cross to grow toproducing seed). Such a method may further comprise (c) crossing a plantgrown from “51-335 RZ”-derived spinach seed with itself or with a secondspinach plant to yield additional “51-335 RZ”-derived spinach seed, (d)growing the additional “51-335 RZ”-derived spinach seed of step (c) toyield additional “51-335 RZ”-derived spinach plants, and (e) repeatingthe crossing and growing of steps (c) and (d) for an additional 3-10generations to further generate “51-335 RZ”-derived spinach plants.Advantageously, a “51-335 RZ”-derived spinach plant has at least thefollowing characteristics: late bolting, savoy mature leaves with anovate shape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410.

Backcrossing one of the parents of a hybrid may also be used to improvean inbred plant. Backcrossing transfers a specific desirable trait fromone inbred or non-inbred source to an inbred that lacks that trait. Thismay be accomplished, for example, by first crossing a superior inbred(A) (recurrent parent) to a donor inbred (non-recurrent parent), whichcarries the appropriate locus or loci for the trait in question. Theprogeny of this cross are then mated back to the superior recurrentparent (A) followed by selection in the resultant progeny for thedesired trait to be transferred from the non-recurrent parent. Afterfive or more backcross generations with selection for the desired trait,the progeny are heterozygous for loci controlling the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The invention additionally provides a method of introducing a desiredtrait into a plant of hybrid spinach variety 51-335 RZ by reversebreeding (See generally U.S. application Ser. No. 10/487,468, publishedas 2006-0179498 A1), which may comprise the following steps: (a)allowing the hybrid spinach plant to produce haploid cells, whilesuppressing recombination, (b) growing haploid plants into diploidplants, (c) selecting those homozygous plants which together constitutethe hybrid variety of the invention as parent plants for the saidhybrid, (d) crossing one of the said parent plants with a plant havingthe desired trait, (e) crossing the selected F1 progeny with said parentplant, to produce backcross progeny; (f) selecting backcross progenywhich may comprise the desired trait and the physiological andmorphological characteristic of the parent plant; and, optionally, (g)repeating steps (e) and (f) one or more times in succession to produceselected fourth or higher backcross progeny that comprise the desiredtrait and all of the physiological and morphological characteristics ofsaid parent plant, (h) crossing the backcrossed parent plant having theadded desired trait with the other parent plant obtained after reversebreeding to obtain a plant which may comprise the desired trait and allof the physiological and morphological characteristics of a plant ofspinach variety 51-335 RZ.

The invention further involves a method of determining the genotype of aplant of spinach variety 51-335 RZ, representative seed of which hasbeen deposited under NCIMB Accession No. NCIMB 41980, or a firstgeneration progeny thereof, which may comprise obtaining a sample ofnucleic acids from said plant and detecting in said nucleic acids aplurality of polymorphisms. This method may additionally comprise thestep of storing the results of detecting the plurality of polymorphismson a computer readable medium. The plurality of polymorphisms areindicative of and/or give rise to the expression of the morphologicaland physiological characteristics of spinach variety 51-335 RZ.

There are various ways of obtaining genotype data from a nucleic acidsample. Genotype data may be gathered which is specific for certainphenotypic traits (e.g. gene sequences), but also patterns of randomgenetic variation may be obtained to construct a so-called DNAfingerprint. Depending on the technique used a fingerprint may beobtained that is unique for hybrid spinach variety 51-335 RZ. Obtaininga unique DNA fingerprint depends on the genetic variation present in avariety and the sensitivity of the fingerprinting technique. A techniqueknown in the art to provide a good fingerprint profile is called theAFLP fingerprinting technique (See generally U.S. Pat. No. 5,874,215),but there are many other marker-based techniques, such as RFLP (orRestriction fragment length polymorphism), SSLP (or Simple sequencelength polymorphism), RAPD (or Random amplification of polymorphic DNA),VNTR (or Variable number tandem repeat), Microsatellite polymorphism,SSR (or Simple sequence repeat), STR (or Short tandem repeat), SFP (orSingle feature polymorphism), DArT (or Diversity Arrays Technology), RADmarkers (or Restriction site associated DNA markers) (e.g. Semagn et al.African Journal of Biotechnology Vol. 5 number 25 pp. 2540-2568, 29Dec., 2006; Baird et al. PloS ONE Vol. 3 number 10 e3376, 2008).Nowadays, sequence-based methods are utilising Single NucleotidePolymorphisms (SNPs) that are randomly distributed across genomes, as acommon tool for genotyping (eg. Elshire et al. PloS ONE Vol. 6 number 5e19379, 2011; Poland et al. PloS ONE Vol. 7 number 2 e32253, 2012;Truong et al. PloS ONE Vol. 7 number 5 e37565, 2012).

With any of the aforementioned genotyping techniques, polymorphisms maybe detected when the genotype and/or sequence of the plant of interestis compared to the genotype and/or sequence of one or more referenceplants. As used herein, the genotype and/or sequence of a referenceplant may be derived from, but is not limited to, any one of thefollowing: parental lines, closely related plant varieties or species,complete genome sequence of a related plant variety or species, or thede novo assembled genome sequence of one or more related plant varietiesor species. For example, it is possible to detect polymorphisms for thecharacteristic of late bolting by comparing the genotype and/or thesequence of hybrid spinach variety 51-335 RZ with the genotype and/orthe sequence of one or more reference plants. The reference plant(s)used for comparison in this example may be any of the standard varietiesFigo, Maracas, Bandola, Viroflay, Matador, Monnopa, Grappa, Medania,Revolver, Chica and Lavewa, Dixie Market, Long Standing Bloomsdale orNorgreen. It is also possible for example, to detect polymorphisms forthe resistance to downy mildew (Peronospora farinosa f. sp. spinaciae)races Pfs1 to Pfs13 and strain UA4410 by comparing the genotype and/orthe sequence of hybrid spinach variety 51-335 RZ with the genotypeand/or the sequence of one or more reference plants. The referenceplant(s) used for comparison may for example be any of the standardvarieties shown in Table 4, or the spinach plant which may comprise theR6 gene, deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen AB21 9YA, UK, on Jul. 26, 2011, under depositaccession number NCIMB 41857.

The polymorphisms revealed by these techniques may be used to establishlinks between genotype and phenotype. The polymorphisms may thus be usedto predict or identify certain phenotypic characteristics, individuals,or even species. The polymorphisms are generally called markers. It iscommon practice for the skilled artisan to apply molecular DNAtechniques for generating polymorphisms and creating markers.

The polymorphisms of this invention may be provided in a variety ofmediums to facilitate use, e.g. a database or computer readable medium,which may also contain descriptive annotations in a form that allows askilled artisan to examine or query the polymorphisms and obtain usefulinformation.

As used herein “database” refers to any representation of retrievablecollected data including computer files such as text files, databasefiles, spreadsheet files and image files, printed tabulations andgraphical representations and combinations of digital and image datacollections. In a preferred aspect of the invention, “database” refersto a memory system that may store computer searchable information.

As used herein, “computer readable media” refers to any medium that maybe read and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM, DRAM, SRAM, SDRAM, ROM; and PROMs(EPROM, EEPROM, Flash EPROM), and hybrids of these categories such asmagnetic/optical storage media. A skilled artisan may readily appreciatehow any of the presently known computer readable mediums may be used tocreate a manufacture which may comprise computer readable medium havingrecorded thereon a polymorphism of the present invention.

As used herein, “recorded” refers to the result of a process for storinginformation in a retrievable database or computer readable medium. Forinstance, a skilled artisan may readily adopt any of the presently knownmethods for recording information on computer readable medium togenerate media which may comprise the polymorphisms of the presentinvention. A variety of data storage structures are available to askilled artisan for creating a computer readable medium where the choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats may be used to store the polymorphisms ofthe present invention on computer readable medium.

The present invention further provides systems, particularlycomputer-based systems, which contain the polymorphisms describedherein. Such systems are designed to identify the polymorphisms of thisinvention. As used herein, “a computer-based system” refers to thehardware, software and memory used to analyze the polymorphisms. Askilled artisan may readily appreciate that any one of the currentlyavailable computer-based system are suitable for use in the presentinvention.

Spinach leaves are processed and sold in packaged form, includingwithout limitation as pre-packaged spinach salad or as canned spinach oras frozen spinach. Such a processing step might comprise but is notlimited to any one of the following treatments or combinations thereof:cutting, washing, blanching, cooking, steaming, baking, pasteurizing,canning, or flash-freezing. Mention is made of U.S. Pat. No. 5,523,136,incorporated herein by reference consistent with the above INCORPORATIONBY REFERENCE section, which provides packaging film, and packages fromsuch packaging film, including such packaging containing leafy produce,and methods for making and using such packaging film and packages, whichare suitable for use with the spinach leaves of the invention. Thus, theinvention comprehends the use of and methods for making and using theleaves of the spinach of the invention, as well as leaves of spinachderived from the invention.

The invention further relates to a container which may comprise one ormore plants of the invention, or one or more spinach plants derived froma plant of the invention, in a growth substrate for harvest of leavesfrom the plant in a domestic environment. This way the consumer may pickvery fresh leaves for use in salads. More generally, the inventionincludes one or more plants of the invention or one or more plantsderived from spinach of the invention, wherein the plant is in aready-to-harvest condition, including with the consumer picking his own,and further including a container which may comprise one or more ofthese plants.

The invention is further described by the following numbered paragraphs:

1. Spinach plant exhibiting a combination of traits including latebolting, savoy mature leaves with an ovate shape, and resistance todowny mildew (Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13and strain UA4410, representative seed of which having been depositedunder NCIMB Accession No. 41980.

2. Spinach plant of paragraph 1 wherein the plant has elliptic firstfoliage leaves.

3. Spinach plant of paragraph 1 or paragraph 2wherein the resistance todowny mildew (P. farinose f. sp. spinaciae) races Pfs1, Pfs2, Pfs3,Pfs4, Pfs5, Pfs6, Pfs9, Pfs11, Pfs12, Pfs13, and isolate UA4410isconferred by the R6 gene.

4. Spinach plant designated 51-335 RZ, representative seed of whichhaving been deposited under NCIMB Accession No. 41980.

5. A seed of the plant of paragraph 1.

6. Parts of the plant of paragraph 1 or paragraph 2 or paragraph 3 orparagraph 4, wherein said parts of the plant are suitable for sexualreproduction.

7. Parts of the plant of paragraph 6, said parts selected from the groupconsisting of microspores, pollen, ovaries, ovules, embryo sacs and eggcells.

8. Parts of the plant of paragraph 1 or paragraph 2 or paragraph 3 orparagraph 4, wherein said parts of the plant are suitable for vegetativereproduction.

9. Parts of the plant of paragraph 8, said parts selected from the groupconsisting of cuttings, roots, stems, cells and protoplasts.

10. A tissue culture of regenerable cells from the spinach plant ofparagraph 1.

11. A tissue culture of paragraph 10, wherein said cells or protoplastsof the tissue culture which are derived from a tissue selected from thegroup consisting of leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds and stems.

12. Progeny of a spinach plant of paragraph 1 or paragraph 2 orparagraph 3 or paragraph 4.

13. Progeny of paragraph 12, wherein said progeny is produced by sexualor vegetative reproduction of said spinach plant, and wherein saidprogeny exhibits a combination of traits including late bolting, savoymature leaves with an ovate shape, and resistance to downy mildew(Peronospora farinosa f.sp. spinaciae) races Pfs1 to Pfs13 and strainUA4410.

14. Progeny of a spinach plant of paragraph 4, having all themorphological and physiological characteristics of the spinach plant ofparagraph 4, representative seed of which having been deposited underNCIMB Accession No. 41980 wherein as found in spinach variety 51-335 RZ,representative seed of which having been deposited under NCIMB AccessionNo. 41980.

15. Progeny of a spinach plant of paragraph 1 or paragraph 2 orparagraph 3 or paragraph 4, representative seed of which having beendeposited under NCIMB Accession 41980, and is modified in one or moreother characteristics.

16. Progeny of paragraph 15, wherein the modification is effected bymutagenesis.

17. Progeny of paragraph 15, wherein the modification is effected bytransformation with a transgene.

18. A method of producing an inbred spinach plant derived from hybridspinach variety 51-335 RZ, comprising the steps:

a) preparing a progeny plant derived from hybrid spinach variety 51-335RZ by crossing the plant of paragraph 1 with a second spinach plant;

b) crossing the progeny plant with itself or a second spinach plant toproduce a seed of a progeny plant of a subsequent generation;

c) growing a progeny plant of a subsequent generation from said seed andcrossing the progeny plant of a subsequent generation with itself or asecond spinach plant; and

d) repeating step b) or c) for at least 1 more generation to produce aninbred spinach plant derived from the hybrid spinach variety 51-335 RZ.

19. The method of paragraph 18 further comprising selecting at steps a),c) and d), an inbred spinach plant exhibiting at least one or more ofthe traits including late bolting, savoy mature leaves with an ovateshape, and resistance to downy mildew (Peronospora farinosa f.sp.spinaciae) races Pfs1 to Pfs13 and strain UA4410.

20. An inbred spinach plant produced by the method of paragraph 18 or19.

21. A method for producing spinach leaves comprising: (a) obtaining aplant according to any one of paragraphs 1-4, wherein the plant has beencultivated to obtain leaves; and (b) harvesting the spinach leaves fromthe plant to thereby obtain harvested spinach leaves.

22. The method of paragraph 21 further comprising packaging theharvested spinach leaves as a fresh vegetable.

23. The method of paragraph 21 further comprising processing theharvested spinach leaves as a processed food.

24. One or more spinach plants of any one of paragraphs 1-4, in acontainer, for the harvest of leaves.

25. A method for producing a seed of a 51-335 RZ-derived spinach plantcomprising (a) crossing a plant of spinach variety 51-335 RZ,representative seed of which having been deposited under NCIMB AccessionNo. NCIMB 41980, with a second spinach plant, and (b) whereby seed of a51-335 RZ-derived spinach plant form.

26. The method of paragraph 25 further comprising (c) crossing a plantgrown from 51-335 RZ-derived spinach seed with itself or with a secondspinach plant to yield additional 51-335 RZ-derived spinach seed, (d)growing the additional 51-335 RZ-derived spinach seed of step (c) toyield additional 51-335 RZ-derived spinach plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional 3-10generations to generate further 51-335 RZ-derived spinach plants.

27. The method of paragraph 25 or 26 further comprising selecting atsteps a), c), and e), a 51-335 RZ-derived spinach plant, exhibiting acombination of traits including late bolting, savoy mature leaves withan ovate shape, and resistance to downy mildew (Peronospora farinosaf.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410.

28. Seed produced by the method of paragraphs 25, or paragraph 26, or27.

29. A method of introducing a desired trait into a plant of hybridspinach variety 51-335 RZ comprising: (a) crossing a parent plant ofhybrid spinach variety 51-335 RZ, with a second pepper plant thatcomprises the desired trait to produce F1 progeny; (b) selecting an F1progeny that comprises the desired trait; (c) crossing the selected F1progeny with said parent plant of spinach variety 51-335 RZ, to producebackcross progeny and (d) selecting backcross progeny comprising thedesired trait and the physiological and morphological characteristic ofsaid parent plant of spinach variety 51-335 RZ, when grown in the sameenvironmental conditions.

30. The method of paragraph 29 wherein the parent plant is obtained byreverse breeding.

31. A method of determining the genotype of a plant of spinach variety51-335 RZ, representative seed of which has been deposited under NCIMBAccession No. NCIMB 41980, or a first generation progeny thereof,comprising obtaining a sample of nucleic acids from said plant andcomparing said nucleic acids to a sample of nucleic acids obtained froma reference plant, and detecting a plurality of polymorphisms betweenthe two nucleic acid samples, wherein the plurality of polymorphisms areindicative of spinach variety 51-335 RZ and/or give rise to theexpression of any one or more, or all, of the morphological andphysiological characteristics of spinach variety 51-335 RZ of paragraph1.

32. The method of paragraph 31 additionally comprising the step ofstoring the results of detecting the plurality of polymorphisms on acomputer readable medium, or transmitting the results of detecting theplurality of polymorphisms.

33. The computer readable medium of paragraph 32.

* * *

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention is not to belimited to particular details set forth in the above description as manyapparent variations thereof are possible without departing from thespirit or scope of the present invention.

What is claimed is:
 1. A spinach plant designated 51-335 RZ, exhibitinga combination of traits including late bolting, savoy mature leaves withan ovate shape, and resistance to downy mildew (Peronospora farinosef.sp. spinaciae) races Pfs1 to Pfs13 and strain UA4410, representativeseed of which having been deposited under NCIMB Accession No.
 41980. 2.A seed of the plant of claim
 1. 3. A part of the plant of claim 1,wherein said part of the plant is suitable for sexual reproduction. 4.The part of the plant as claimed in claim 3, wherein said part comprisesa microspore, pollen, ovary, ovule, embryo sac or egg cell.
 5. A part ofthe plant of claim 1, wherein said part of the plant is suitable forvegetative reproduction.
 6. The part of the plant as claimed in claim 5,wherein said part comprises a cutting, root, stem, cell or protoplast.7. A tissue culture of regenerable cells or protoplasts from the spinachplant claim
 1. 8. The tissue culture as claimed in claim 7, wherein saidcells or protoplasts of the tissue culture are derived from a tissuecomprising a leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell, root, root tip, anther, flower, seed or stem.
 9. A spinach plantproduced by transforming the spinach plant of claim 1 with a transgene.10. A method of producing an inbred spinach plant derived from hybridspinach variety 51-335 RZ, comprising the steps: a) preparing a progenyplant derived from hybrid spinach variety 51-335 RZ by crossing theplant of claim 1 with a second spinach plant; b) crossing the progenyplant with itself or a second spinach plant to produce a seed of aprogeny plant of a subsequent generation; c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second spinach plant; and d)repeating step b) and c) for at least 1 more generation to produce aninbred spinach plant derived from hybrid spinach variety 51-335 RZ. 11.The method of claim 10 further comprising selecting at steps a), c) andd), an inbred spinach plant exhibiting at least one or more of thetraits including late bolting, savoy mature leaves with an ovate shape,and resistance to downy mildew (Peronospora farinose f.sp. spinaciae)races Pfs1 to Pfs 13and strain UA
 4410. 12. A method for producingspinach leaves comprising: (a) obtaining a plant according to claim 1,wherein the plant has been cultivated to obtain leaves; and (b)harvesting the spinach leaves from the plant to thereby obtain harvestedspinach leaves.
 13. The method of claim 12 further comprising packagingthe harvested spinach leaves as a fresh vegetable.
 14. The method ofclaim 12 further comprising processing the harvested spinach leaves as aprocessed food.
 15. A container comprising one or more spinach plantsclaim 1 for the harvest of leaves.
 16. A method for producing a seed ofa 51-335 RZ-derived spinach plant comprising (a) crossing a plant ofspinach variety 51-335 RZ, representative seed of which having beendeposited under NCIMB Accession No. 41980, with a second spinach plant,and (b) whereby seed of a 51-335 RZ-derived spinach plant forms.
 17. Themethod of claim 16 further comprising (c) crossing a plant grown from51-335 RZ-derived spinach seed with itself or with a second spinachplant to yield additional 51-335 RZ-derived spinach seed, (d) growingthe additional 51-335 RZ-derived spinach seed of step (c) to yieldadditional 51-335 RZ-derived spinach plants, and (e) repeating thecrossing and growing of steps (c) and (d) for an additional3-10generations to generate further 51-335 RZ-derived spinach plants.18. The method of claim 17 further comprising selecting at steps a), c),and e), a 51-335 RZ-derived spinach plant, exhibiting a combination oftraits including late bolting, savoy mature leaves with an ovate shape,and resistance to downy mildew (Peronospora farinose fsp. spinaciae)races Pfs1 to Pfs13and strain UA4410.
 19. A method of determining thegenotype of a plant of spinach variety 51-335 RZ, representative seed ofwhich has been deposited under NCIMB Accession No. 41980, or a firstgeneration progeny thereof, comprising obtaining a sample of nucleicacids from said plant and comparing said nucleic acids to a sample ofnucleic acids obtained from a reference plant, and detecting a pluralityof polymorphisms between the two nucleic acid samples, wherein theplurality of polymorphisms are indicative of spinach variety 51-335RZand/or give rise to the expression of any one or more, or all, of themorphological and physiological characteristics of spinach variety51-335 RZ as claimed in claim
 1. 20. The method of claim 19 additionallycomprising the step of storing the results of detecting the plurality ofpolymorphisms on a computer readable medium, or transmitting the resultsof detecting the plurality of polymorphisms.
 21. The plant of claim 1,which is a plant grown from seed having been deposited under NCIMBAccession No.
 41980. 22. A spinach plant having all the morphologicaland physiological characteristics of a plant as claimed in claim 1,representative seed of which having been deposited under NCIMB AccessionNo.
 41980. 23. A seed capable of growing into a plant of claim 1.